Project Summary/Abstract Alcohol use disorder (AUDs) is a highly prevalent substance use disorder that affects more than 16 million Americans and poses a serious health and socioeconomic burden upon the unwell and their environment. The only FDA-approved treatments are only modestly effective, causing aversive side-effects or requiring active and continued abstinence. AUDs are characterized at a molecular level by long-lasting changes in gene expression, which can be controlled in part by epigenetic machinery. 5-methylcytosine (5mC) and the more recently discovered 5-hydroxymethylcytosine (5hmC) are stable epigenetic modifications of the DNA itself that play large roles in regulating gene expression. It has long been known that 5mC and 5hmC are involved in diseases such as cancer. Recently, 5mC and 5hmC and the enzymes that create them (DNMT and TET proteins, respectively) have been shown to play a role in cocaine abuse and DNMTs have been implicated in AUD, but no research has been published on the role of 5hmC in the context of AUD. The proposed research will investigate the relationship between chronic alcohol and DNA modifications. The first aim will break down the relationship between changes in the DNA modification system and chronic intoxication and withdrawal by characterizing this epigenetic system in the prefrontal cortex, which undergoes changes to gene expression and normal function after chronic (but not acute) alcohol use. We will use the chronic intermittent ethanol vapor model of alcohol intoxication, dependence, and withdrawal to treat C57BL/6J mice. DNA-level epigenetic regulatory systems will be probed at RNA, protein, and DNA levels to determine expression of DNMT and TET genes and proteins and global 5mC and 5hmC content, respectively. The second aim will clarify the role of the TET1 protein and 5hmC in the context of AUDs by virally manipulating Tet1 expression to determine its role in regulating post-vapor induced dependence drinking behaviors through 5hmC. This project will expand the growing body of knowledge regarding the important regulatory role of epigenetic factors in substance use disorders, specifically with respect to the unexplored role of 5hmC, and may serve to identify new potential targets for the treatment of AUD. DNMT inhibitors have been shown to alter drinking behaviors, and between those findings and the identification of a role for TET1 and 5hmC in cocaine behaviors, it stands to reason that 5hmC may be involved in regulating alcohol behaviors. The proposed research will clarify the link between alcohol use and DNA-level epigenetic regulation, and will be key in bringing DNA modifications to light as a potential therapeutic target for treatment of alcohol and other substance use disorders. Furthermore, the findings of the proposed research may have implications for the treatment of other psychiatric disorders, especially those that have co-morbidities with AUD and have been shown to have disrupted DNA-level epigenetic regulatory systems, like schizophrenia and bipolar disorder.